Defensins are small cationic peptides with conserved structure, which are secreted by a variety of cells and have anti-viral, anti-bacterial and anti-fungal activity. Alpha, beta and theta defensins all have activity against HIV- 1; together they may constitute an important component of innate immunity to HIV-1. Defensins secreted in the gut, female reproductive tract, oral cavity and blood may all play a role in combating HIV-1. Despite numerous reports of anti-HIV- 1 activity of defensins, little is know about their mechanisms of action. Insight into these mechanisms will increase our knowledge about the replication of HIV- 1 in vivo and may allow us to augment natural defensins with administered defensins or defensin-like drugs that could be used systemically or topically. To achieve these goals we will identify the most potent ant-HIV-1 defensins and investigate their mechanisms of action. Our specific aims are: 1. To assay the anti-HIV-1 activity of all known human and selected non-human defensins. Human alpoha and beta defensins and rhesus macaque 0 defensins will be produced in E. coli or chemically synthesized and purified by HPLC. Defensins will be added to HIV- 1 in low salt buffer or to cells in serum-flee medium. GHOST cells, PBMC, fetal thymic organ culture and lymphoid tissue histoculture, will be used to evaluate defensin mediated inhibition of R5 and X4 HI-V- 1 replication. 2. To characterize the mechanism by which each active defensin inhibits HIV-1 replication. Defensins will be added to cells or virus, before infection and to cells during and after infection to determine the site and time of action. Defensin binding to virus and cells will be assayed with radiolabeled defensins and confirmed with anti-defensin antibodies when possible. The stage at which viral replication is inhibited will be determined by quantitative PCR for viral DNA and RNA and assays of viral proteins and infectivity.